Cell Division Course Lecturers 2014

Full Name

Phone

Pumin Zhang

713-798-1866 BCM T411

pzhang@bcm.edu

Anna Marie Sokac

713-798-3561 BCM S307

sokac@bcm.edu

Eric Chang

713-798-3519 BCM
N1110.01
713-798-1565 N803.6

echang1@bcm.edu

Weiwei Dang

Room

e-mail

Weiwei.Dang@bcm.edu

Alison Bertuch

832-824-4579 Feigin Center aabertuc@txch.org

1240.08

Thomas Westbrook

713-798-5364 BCM 304E

thomasw@bcm.edu

Greg Ira

713-798-1017 BCM T817

gira@bcm.edu

TAs 2014
Emily Dawson
Emily.Dawson@bcm.edu
Jarrett Kelsey
Kelsey.Jarrett@bcm.edu

Class Schedule
Exams (2)
First 7 lectures (70 points)
Second 5 lectures (50 points)
(10 points per lecture)
TA Sessions (4)
Run by TAs
Not obligatory
Emphasis on lectures and assigned readings
E-mail questions in advance
2 Faculty Review Sessions

Cell Division Course Readings

Course textbooks:
General material: Molecular Biology of
the Cell (Alberts)
Cell cycle:

The Cell Cycle, Principles of

Control (Morgan)

Lecture-specific references:
Assigned and posted* by each lecturer
(*or, students download from PubMed)

CELL DIVISION LECTURE 1 OUTLINE

Pumin Zhang October 07, 2013

basic principles and the big picture of cell cycle

(context for the future lectures)

DNA, Cell, Life

Life:

Reproduction

Cell:

Unit of life

DNA:

Genetic information storage

Cell Division Cycle

Cell cycle under a microscope

Molecular Biology of the Cell, 4th Edition

Based on early microscope studies, cells were found to

have two morphologically distinct cell cycle states:
Interphase and Mitosis (M phase)

Gap1

Gap2

Molecular Biology of the Cell, 4th Edition

Interphase was later

found to consist of three
distinct phases:

G1 (prior to replication)
S (DNA Synthesis)
G2 (after replication)

Mitosis vs. Meiosis

The distribution of cells in the cell cycle varies

according to cell type and organism

Figure 17-4 Molecular Biology of the Cell ( Garland Science 2008)

The distribution of cells in the cell cycle varies

according to cell type and organism

2006 New Science Press Ltd new-science-press.com

The cell cycle consists of a series of orderly, periodical events

How are these events coordinated?

What governs which happens first?

Johnson and Rao cell fusion experiments the concept

Johnson & Rao Nature (1970) 226:717

Evidence for the existence of

MPF (Mitosis Promoting Factor)
and SPF (S phase Promoting Factor)

Xenopus Eggs Are Rich Sources of Cell Cycle Regulators

Fully Grown
Oocyte
(G2 arrest)

Completion
of Meiosis I

MPF Activity

Progesterone

Interphase
arrest

Meiosis II
Arrest

Fertilized
Egg

First
Mitosis

Second
Mitosis

Fertilization

Metaphase
arrest

Masui & Markert (1971)

Discovery of MPF in frog oocytes the experimental strategy

Inject cytoplasm
from M-phase cell

Oocyte

spindle
Oocyte is
driven into M
phase

Inject cytoplasm from

interphase cell

Oocyte

Oocyte
remains in G2
Masui & Markert (1971)

Purification of MPF
One Component is CDC2

cyclin
CDC2
(now called
CDK1)
H1 kinase
activity

(in extract)

(oocytes)

(silver stained)

PNAS (1988) 85:3009

Purification of MPF

What is Second Component?

H1 kinase
activity

(in extract)

(oocytes)

(B&C: autoradiograph)

PNAS (1988) 85:3009

Cell (1990) 60:487

Examination of new protein synthesis in egg extracts

by Tim Hunt identified another key cell cycle regulator:
cyclin

[35S] methioninelabeled sea urchin

egg extracts
analyzed by SDSPAGE

cyclin

Cell. 1983 Jun;33(2):389-96.

Purification of MPF

Second Component is Cyclin

cyclin

(in extract)

(oocytes)

H1 kinase
activity

PNAS (1988) 85:3009
Cell (1990) 60:487

MPF = Kinase + Cyclin

Isolation of temperature sensitive yeast cell division cycle (cdc)

mutants was key to identifying critical cell cycle regulatory genes

Isolating mutants in
a large population

25oC

36oC
Figure 8-55 Molecular Biology of the Cell ( Garland Science 2008)

Temperature sensitive
Cloning
genes by functional
Temperature
mutant
cells transformed
sensitive
with
gene library
complementation

mutant cells
transformed
with gene library

Mutant cells transformed

with gene library
plasmids
Modified from Figure 8-55 Molecular Biology of the Cell ( Garland Science 2008)

All cells with library

plasmid proliferate and
form a colony at the
permissive temperature

25oC

36oC

Cells carrying a plasmid

with wild type copy of
gene proliferate and
form a colony at the
non-permissive
temperature

Hartwell (1971); Nurse, Thuriaux & Nasmyth (1976)

Yeast cell division cycle (cdc) mutants

the genetic approach
S phase

G2
S phase
G1

M phase

S phase
G2

M phase
G1

Schizosaccharomyces pombe fission yeast

Long G2, short G1
Paul Nurse:
Isolated cdc mutants based on cell elongation.

Saccharomyces cerevisiae budding yeast

Long G1, short G2
Lee Hartwell:
Isolated cdc mutants based on cell morphology

Assessed DNA content and nuclear morphology

to determine arrest point in cell cycle.

Assessed DNA content and nuclear morphology

to determine arrest point in cell cycle.

Evolutionarily, fission yeast and budding yeast are nearly as

divergent from one another as either is from humans

Isolation of temperature sensitive yeast cell division cycle (cdc)

mutants was key to identifying critical cell cycle regulatory genes

Isolating mutants in
a large population

25oC

36oC
Figure 8-55 Molecular Biology of the Cell ( Garland Science 2008)

Temperature sensitive
Cloning
genes by functional
Temperature
mutant
cells transformed
sensitive
with
gene library
complementation

mutant cells
transformed
with gene library

Mutant cells transformed

with gene library
plasmids
Modified from Figure 8-55 Molecular Biology of the Cell ( Garland Science 2008)

All cells with library

plasmid proliferate and
form a colony at the
permissive temperature

25oC

36oC

Cells carrying a plasmid

with wild type copy of
gene proliferate and
form a colony at the
non-permissive
temperature

Hartwell (1971); Nurse, Thuriaux & Nasmyth (1976)

cdc:
1. cell cycle regulators
2. essential for a particular event,
for example DNA synthesis.

Examples: cdc2 & cdc25

mutants. Later found to
be blocked in G2

S!

G1!
M!

G2!

Figure 17-6 Molecular Biology of the Cell ( Garland Science 2008)

S!
G1!
M!

G2!

Modified from: Figure 17-6 Molecular Biology

of the Cell ( Garland Science 2008)

Genetic Analysis Deciphered the

Basic Cell Cycle Regulatory Network
Protein
kinase

G2

Protein
phosphatase

wee1 cdc25

Cdc2
Inactive

Cdc2
Active

Protein kinase

Protein kinase

cdc2ts Mutant
wee1 OE

cdc2-w Mutant
wee1 Mutant

cdc25ts Mutant

cdc25 OE

Genetic Analysis Deciphered the

Basic Cell Cycle Regulatory Network
Protein
kinase

G2

Protein
phosphatase

wee1 cdc25

Cdc2
Inactive

Cdc2
Active

Protein kinase

Protein kinase

Even more strikingly, cdc2 is also required for S phase entry.

BUT Mitosis and DNA synthesis are two very different events !!

Isolation of temperature sensitive yeast cell division cycle (cdc)

mutants was key to identifying critical cell cycle regulatory genes

Isolating mutants in
a large population

25oC

36oC
Figure 8-55 Molecular Biology of the Cell ( Garland Science 2008)

Temperature sensitive
Cloning
genes by functional
Temperature
mutant
cells transformed
sensitive
with
gene library
complementation

mutant cells
transformed
with gene library

Mutant cells transformed

with gene library
plasmids
Modified from Figure 8-55 Molecular Biology of the Cell ( Garland Science 2008)

All cells with library

plasmid proliferate and
form a colony at the
permissive temperature

25oC

36oC

Cells carrying a plasmid

with wild type copy of
gene proliferate and
form a colony at the
non-permissive
temperature

Hartwell (1971); Nurse, Thuriaux & Nasmyth (1976)

Universal control of the cell cycle?

Cloning of human CDC2 gene

by complementation of
fission yeast cdc2ts mutant

Cloning human CDC 2 gene
Temperature sensitive
Temperature
mutant
cells transformed
sensitive
with gene library

mutant cells
transformed
with gene library

Yeast mutant cells

transformed with
human gene
library plasmids

25oC

36oC

Lee & Nurse (1987)

Cyclins are the regulatory subunits of the CDK class of

kinases more in my next lecture

2006 New Science Press Ltd new-science-press.com

1) Activate CDK- essential for activity

2) Sometimes help to recruit substrates

3) Govern the duration of the CDK active state

4) Target CDK to subcellular location

The Nobel Prize in Physiology or Medicine

2001 for work on the cell cycle

Leland H. Hartwell
USA

R. Timothy (Tim) Hunt Sir Paul M. Nurse

United Kingdom
United Kingdom

Fred Hutchinson
Cancer Research Center
Seattle, WA, USA
b. 1939

Imperial Cancer
Research Fund
London, United Kingdom
b. 1943

President,
Rockefeller University
New York, USA
b. 1949

Assigned readings for this lecture:

Nurse, P. (2000) A long twentieth century of the cell cycle.
Cell 100:71-78
Nasmyth, K. (2001) Prize for Proliferation.
Cell 107: 689701.

The cell cycle consists of a series of orderly, periodical events

Analyze a cell population by FACS

Fluorescence Activated Cell Sorter (FACS) !
also called Flow Cytometer!

Figure 8-2 Molecular Biology of the Cell ( Garland Science 2008)

Flow Cytometry Used to Measure Relative DNA

Content of Cells Stained with Fluorescent DNA Binding Dye!
FACS in essence provides a
snapshot of the composition
of a population.
Implications:
1.Determine proportion of
cells in each phase (area
under the peak)
2. Determine the relative
length of each phase of the
cell cycle
3. Determine the absolute
length of each phase

Flow Cytometry can be used to estimate the proportion of

cells in each cell cycle phase!
Example:
In this histogram the
number of cells in G1 is
approximately three times
the number in G2+M. The
number in S is
approximately equal to
that of G2+M
Conclusion:
G1 is ~3/5 of the cycle
G2+M is ~1/5 of the cycle
S is ~1/5 of the cycle

Flow Cytometry can be used to estimate the length

of each cell cycle phase!
Example:
In this histogram of an
asynchronously growing
population, ~3/5 of the
cells are in G1.
Conclusion:
If you know that the
doubling time of the
population is 20 hours you
can calculate the length of
the G1 phase
Hint:
20 hours X 3/5

number of cells

Flow cytometry can be used to monitor

cell cycle progression in a synchronous population!

2007 New Science Press Ltd new-science-press.com

Fidelity of cell division:

-Importance of a high fidelity:
The vast scales of cell division;
Severe consequence of errors.
-The fidelity of each stage, and individual steps within,
contributes to the overall fidelity of cell division.
DNA replication;
Chromatin replication (epigenetic factors);
Chromosome segregation
-The remedies:
DNA damage repair;
Checkpoints;
Apoptosis

Assigned Readings